2.5 Restriction Mapping
2.5 Restriction Mapping margaret Wed, 11/16/2011 - 18:16W 2/22/12
Goal for this lab: Purify the amplified DNA from first lab, digest it with restriction enzymes, and assess the results with gel electrophoresis.
Zymo Research DNA Clean & Concentrator-5 Cat # T1225 K
$69 for kit of 50
Reagents Per Pair:
- 125 µL Zymo-SpinTM DNA Binding Buffer
- 500 µL Zymo-SpinTM Wash Buffer (GET MORE FOR 2015)
- 1 mL Sterile ddH2O
- 1 Zymo-Spin™ Column
- 2 Capless collection tubes
10 µL 20 mM Spermidine [Should this be 3 tubes of 1 uL??]
1x TAE for 14 gels @ 300 mL/gel = 4200 mL (make at least 5 L)
- 1 2% gel per pair plus 2
- 10x NEB restriction buffer. Students need at most 10µL of any 1 buffer. Check supplies
- Fermentas MassRuler, Low Range
Restriction Enzymes for 2012
Grp | enz 1 | enz 2 | NEB buffer |
---|---|---|---|
ASP | Xho1 | Hind3 | 2 |
BLW | EcoRV | Cla1 | 3, 4, (2) |
CHC | Bgl2 | Cla1 | 3, 4 |
CNT | pvu1 | EcoRV | 3 |
EFO | Hpa1 | Mlu1 | 4, 3 |
KSY | Spe1 | Sph1 | 2 |
LSO | Sal1 | EcoRV | 3 |
NOD | EcoRV | Sac1 | 1, 2, 3, 4 |
RFR | Cla1 | Hind3 | 4,2, |
RMT | Bgl2 | Pvu2 | 3 |
Bgl2 Cla1 EcoRV Hind3 Hpa1 Mlu1 pvu1 Pvu2 Sac1 Sal1 Spe1 Sph1 Xho1
Equipment
- Incubator at 37C, with microtube racks inside
- Water bath at 65C for gels
- copies of semi-log paper (1 per student) (see attached document)
- find powerpoint of semi-log paper and put it on the course website
2014 RE
2014 RE kdorfman Wed, 02/19/2014 - 20:04MW | TuTh | buf1 | buf 2 | buf 3 | buf 4 | cutsmart |
---|---|---|---|---|---|---|
AatII | Aat II | 0 | 50 | 50 | 100 | 100 |
AclI | AclI | 100 | ||||
AflII | AflII | 50 | 100 | 25 | 100 | 100 |
BciVI | 100 | |||||
Bgl2 | 10 | 75 | 100 | 10 | 10 | |
BmgBI | 3.1-100 | 10 | ||||
BspE1 | 0 | 10 | 100 | 0 | 10 | |
BsrBI | 100 | |||||
BsrGI | 25 | |||||
ClaI | 10 | 50 | 50 | 100 | 100 | |
DraI | 100 | |||||
EarI | 100 | |||||
EcoRI | 100 | 100 | 100 | 100 | 50 | |
EcoRV | 50 | 75 | 100 | 50 | 10 | |
HindIII | HindIII | 50 | 100 | 10 | 50 | 50 |
HpaI | 25 | 50 | 10 | 100 | 100 | |
MluI | 25 | 75 | 100 | 50 | 25 | |
NcoI | 100 | 100 | 100 | 100 | 100 | |
Pvu2 | 100 | 100 | 100 | 100 | 100 | |
SexAI | 100 | |||||
SpeI | 75 | 100 | 25 | 75 | 100 | |
XbaI | 0 | 100 | 75 | 75 | 100 |